Butylated hydroxytoluene (BHT) is a potent inactivator of the enveloped bacterial virus 46 at concentrations as low as 3 x 10-5 M. The viral envelope is not removed by BHT treatment, in contrast to the effects of exposure to the detergent Triton X-100. BHT-treated viruses are morphologically indistinguishable from controls but are defective in their ability to attach to the host cell. Temperature at the time of exposure was found to .be a crucial factor in the effectiveness of BHT against gb6. A precipitous drop in the degree of inactivation by 3 x 10-5 M BHT occurred when the temperature was lowered from 20 to 15 C. Calcium ions were found to potentiate the effect of BHT, particularly at lower temperatures where BHT alone was relatively ineffective. Barium and strontium, but not magnesium, were also effective in enhancing the activity of BHT. A structurally related molecule, butylated hydroxyanisole (BHA), was also found to inactivate 46 virus, but higher concentrations were required than with BHT. Both BHT and BHA are commonly used as food additives, have apparent low toxicity to humans and other animals, and are potentially useful as antiviral agents.It was recently reported from our laboratory that butylated hydroxytoluene (BHT) is a potent inactivator of lipid-containing viruses (11). This compound is generally recognized as safe by the Food and Drug Administration (5) and is widely used as an antioxidant in food and cosmetic preparations. The combination of effectiveness against viruses and apparent low toxicity to humans has generated considerable interest in the potential use of BHT as an antiviral agent, particularly for topical application to virus-infected areas of the skin. Consequently, we are characterizing the effects of BHT on lipid-containing viruses in vitro, hoping to improve our understanding of the mechanisms whereby BHT destroys viral infectivity.In a detailed study of the effects of BHT on the bacterial virus PM2, it was found that virus particles are completely disrupted by BHT, with the viral deoxyribonucleic acid being released to the medium as a very slowly sedimentable material (1). Several factors which influence the inactivation of PM2 by BHT, including the initial virus titer, the time of exposure, and the presence of certain "protective" agents including surfactants, bovine serum al- ' Present address: Washington University Medical School, Cancer Center, St. Louis, Mo. 63130. bumin, and bacterial cells, were studied. Potentially important with regard to the use of BHT as an antiviral substance was the observation that its effectiveness is strongly dependent upon the solvent from which it is added to the culture medium.Different viruses have different susceptibilities to BHT, and the most susceptible one studied thus far is the enveloped bacterial virus 46. This virus is bounded by a rather loose membrane structure (3, 13) and has many similarities to several animal viruses. We have now studied in more detail the inactivation of46 by BHT, and the results of this research are de...

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Communicated by Oliver H. Lowry, September 7, 1979 ABSTRACT Rough and smooth microsomes were prepared from ascites tumor cells, rat liver, and bovine adrenal cortex. Proteolytic removal of the signal peptide in pre-placental lactogen and asparagine-linked glycosylation of the a subunit of

Ribosomes unfolded by the removal of Mg2+ at 25 C were studied by Raman spectroscopy and electron microscopy. Raman spectra showed a reduction in the 813 cm-l phosphodiester signal of 30S and 50S ribosomes compared to intact ribosomes, suggesting that a fraction of the ribose moieties had shifted from the 3' endo (ordered) to the 3' exo (disordered) conformation.The maximum diameters of unfolded 30S and 50S ribosomes, judged by electron microscopy, were 1.8 and 2.5-fold greater, respectively, than those of intact ribosomes. Most unfolded 30S ribosomes had three distinct structural domains and appeared "Y-shaped"; whereas most unfolded 50S ribosomes had four distinct domains and appeared "X-shaped". When ribosomes were partially unfolded (by brief exposure to 0.04 mM Mg2+ or EDTA), several possible intermediates in the unfolding process were observed.Both the shapes of particles and their Raman spectra reached the same final state in 0.04 mM Mg2+, where more than 50% of the rRNA phosphates are discharged by Mg2+, as in 10 mM EDTA, where less than 1% are discharged.

INTRODUCTIONThe endoplasmic reticulum (ER) is intimately involved in several processing events that are associated with synthesis and transport of eukaryotic secretory proteins. For example, the prepeptides, located at the amino termini of nascent polypeptide chains are removed during their translation by a n activity present in microsomal membranes,l resulting in the sequestration of the cleaved proteins within these membranes. Moreover, the dolichol-bound oligosaccharide core, which is transferred t o specific asparagine residues in newly synthesized glycoprotein chains, is located in the endoplasmic reticulum.' This initial oligosaccharide core is rich in mannose and undergoes one or more obligatory trimming reactions before further coupling of sugars unique to specific glycoproteins is seen.' Presumably these reactions occur later in the secretory pathway through the smooth endoplasmic reticulum and Golgi fractions.Previously we reported that in lysates supplemented with microsomal membranes derived from ascites tumor cells, human placental mRNA directed the synthesis of a mannose-rich glycosylated form of the alpha subunit of human chorionic gonadotropin (hCG-a)'. To study processing of this oligosaccharide, we prepared subcellular fractions from ascites tumor cells and rate liver. Unexpectedly, we observed that smooth microsomal fractions effectively sequestered and glycosylated nascent chains of hCG-a. In addition, smooth microsomes also processed the preform of the nonglycosylated protein preplacental lactogen (pre-hPL).It has been suggested that the major distinction between the rough and smooth endoplasmic reticulum is the absence in the latter of specific ribosomal binding sites. The results presented here strongly suggest that in lysates from ascites tumor cells, smooth microsomes are nevertheless capable of interacting with polysomes bearing placental pre-peptide nascent chains. MATERIALS AND METHODSAntiserum generated against highly purified, reduced, and carboxymethylated hCG-a was prepared by S. Birken and R. Canfield as previously described.' a-1-Antitrypsin inhibitor was obtained from Sigma. * This work was supported by grants from the National Institutes of Health (PO-HD-08235) and the Population Council. M.B. is a Ford Foundation Fellow. 69 0077-8923/80/03434069 501.75/0 0 1980, NYAS 70 Annals New York Academy of SciencesFIGURE 1 . Endoplasmic reticulum (ER) fractions from ascites tumor cells and liver cells. (A) Smooth ER from ascites cells. Note A-Type viral particles (arrows). (B) Smooth ER from liver cells. C and D correspond to tumor and liver rough fraction, respectively. Sections were double stained with alcoholic uranyl acetate and lead citrate." Marker bars represent 100 nm. Preparation of Subcellular FractionsPlacental RNA, ascites tumor ribosomes and cell-sap (S-100) were prepared as previously described.' Assay of protein synthesis, immunoprecipitation of the cell-Boime et al. : Processing of Placental Peptide Hormones 71 free products, and their resolution on sodium dodecyl sulfat...